Method on clinical applications in head neck cancer by using DSG3 molecule for predicting malignant degree of cancer, serving as a molecular target and using RNA jamming sequence on inhibition-specific of DSG3 expression

ABSTRACT

The present invention provide a method for analyzing the DSG3 overexpression in tumor tissues with clinical features of cancer cells to validate that overexpression is relates to size, depth and migration of tumor. Therefore, DSG3 overexpression is capable for using in clinical applications, determining malignant degree of tumor, serving as molecular target in Head Neck Cancer (HNC). Moreover, a jamming sequence, RNA, is designed to act on DSG3 mRNA and is effective inhibition-specific DSG3 expression, and then inhibits cell growth, invasion and migration in HNC.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a division of U.S. patent application Ser.No. 11/593,435, filed on Nov. 6, 2006 (now U.S. Pat. No. 7,652,456),titled Method on Clinical Applications in Head Neck Cancer by Using DSG3Molecule for Predicting Malignant Degree of Cancer, Serving as aMolecular Target and Using RNA Jamming Sequence on Inhibition-Specificof DSG3 Expression, listing Joseph Tung-Chieh Chang, Ann-Joy Cheng andYin-Ju Chen as inventors.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a DSG3 molecule that is identified therelation between the overexpression of DSG3 and the malignant degree ofclinical head neck cancer (HNC), and more particularly to a method forproviding a RNA jamming sequence on inhibition-specific of DSG3expression to inhibit the growth, invasion and migration of cancercells.

2. Description of Related Art

Head Neck Cancer (HNC) is threatening the human health seriously, thetendency on suffering from HNC is increasing year by year, and theresearch of HNC on the cause, diagnosis and prognosis is a veryimportant topic therefore. There is no conventional technology by usingthe overexpression or underexpression of cell molecule to determine themalignant degree of cancer or be the molecular-target on the therapy ofHNC.

SUMMARY OF THE INVENTION

The present invention investigates and finds a DSG3 molecule relative tothe HNC to study the DSG3 expression of normal and tumor tissues from 56patients with HNC, and to identify the DSG3 overexpression is relativeto the malignant degree of clinical HNC that is including the size anddepth of cancer and Lymph metastasis. Therefore, the DSG3 expression iscapable of determining the malignant degree of cancer on clinicalapplications and being a molecular target on the therapy of HNC.

The present invention also provides a method by using a RNA jammingsequence on inhibition-specific of DSG3 expression, and moreparticularly to a RNA jamming sequence on inhibition-specific of DSG3mRNA expression to be identified on inhibition-specific of DSG3 proteinexpression effectively and then inhibit the growth, invasion andmigration of cancer cells.

Further benefits and advantages of the present invention will becomeapparent after a careful reading of the detailed description withappropriate reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a diagram of Gel Electrophoresis of DSG3 RNAoverexpression in tumor tissue that DSG3 RNA expression as determined byPT-PCR in sample of HNC.

FIG. 1B shows a diagram of Gel Electrophoresis of DSG3 proteinoverexpression in tumor tissue that DSG3 protein expression asdetermined by Western blot analysis in sample of HNC.

FIG. 2 shows a diagram of hairpin structure by DSG3 RNA interference inHNC (SEQ. ID. NO. 6).

FIG. 3A shows a diagram on effects of DSG3-RNAi on DSG3 proteinexpression in OECM1 cells, and cells are transfected with DSG-RNAi andfurther cultured for 4 days. FIG. 3B shows a diagram on effects ofDSG3-RNAi on DSG3 protein expression in BM1 cells, and cells aretransfected with DSG-RNAi and further cultured for 4 days.

FIG. 4A shows a diagram that DSG3 knockdown inhibits cell growth inOECM1 cell lines, and cells are transfected with DSG-RNAi and furthercultured for 4 days.

FIG. 4B shows a diagram that DSG3 knockdown inhibits cell growth in BM1cell lines, and cells are transfected with DSG-RNAi and further culturedfor 4 days.

FIG. 5A shows a diagram of cell analysis to indicate that DSG3 knockdowninhibits colony formation in OECM1 and BM1 cell lines.

FIG. 5B shows a diagram on quantitative determination of colony numbersto indicate that DSG3 knockdown inhibits colony formation in OECM1 andBM1 cell lines.

FIG. 6 shows a diagram in vitro wounded healing assay to indicate thatDSG3 knockdown reduces cell migration in OECM1 and BM1 cell lines.

FIG. 7A shows a diagram in vitro Matrigel Transwell invasion assays toindicate that DSG3 knockdown reduces cell migration in OECM1 cell lines.

FIG. 7B shows a diagram in vitro Matrigel Transwell invasion assays toindicate that DSG3 knockdown reduces cell migration in BM1 cell lines.

FIG. 8A shows a diagram of effect of DSG3-RNAi on tumor xenograftBALB-C/nu mice to validate that DSG3 Knockdown inhibits cancer cellgrowth in vivo.

FIG. 8B shows a diagram of immunohistochemical (IHC) analysis tovalidate that DSG3 expression is certainly inhibited by DSG3-RNAi

FIG. 9A shows a diagram of liver organ to validate that DSG3 Knockdowninhibits cancer migration in vivo.

FIG. 9B shows a diagram of statistical table to indicate that DSG3Knockdown inhibits cancer migration in vivo

FIG. 10 shows a diagram of characteristics of HNC patients recruited inthis study.

FIG. 11 shows a diagram on DSG3 expression with clinicopathologicstatus.

DETAILED DESCRIPTION OF THE INVENTION A. Relation Between DSG3Expression and Clinical Malignant Degree in HNC

1. DSG3 Overexpression at Both RNA and Protein Levels

The present invention analyses the normal and cancerous tissues toidentify the DSG3 expressions at both RNA and protein levels. At RNAlevel, a specific sequence of DSG3 is enlarged by using ReverseTranscription-Polymerase Chain Reaction (PT-PCR) and is analyzed by GelElectrophoresis (as shown in FIG. 1A), and the analysis at protein leveluses Western blot analysis (as shown in FIG. 1B), wherein N 10 is normaltissue, T 20 is tumor tissue, 18S 30 is the expression of RNA in samplethat is able to be an internal control of RNA expression, and Actin 40is cytoskeletal protein expression that is able to be the internalcontrol of protein expression. Regardless the RNA or protein level, DSG3expression is higher in the tumor tissues than in the normal tissuesfrom the same patient in the figures.

2. Method for Analyzing the Difference Between Normal and Tumor Tissuesfrom 56 Patients with HNC

Furthermore, normal and tumor tissues from 56 patients with HNC wereobtained and analyzed by real time PT-PCR to identify the DSG3expression, and the characteristics of these HNC patients weresummarized in the FIG. 10. The patients included 52 (93%) males and four(7%) females, with an age range of 32-74 years (median 51 years),wherein 10 younger than 40 years old, 17 between 41 and 50 years old, 22between 51 and 60 years old, 6 between 61 and 70 years old, and 1 olderthan 70 years old. A total of 44 (79%) consumed alcohol, 51 (91%) smokedtobacco and 50 (89%) chewed betel nut. The percentage of having thehabits of smoking, drinking and chewing betel nut is about 80% in totalpatients.

The relative expression of DSG3 from each tumor sample compared withthat from the normal tissue from the same patient is defined after theDSG3 RNA expression of each sample is normalized with an internalcontrol (18S RNA expression). DSG3 expression in tumor tissue greaterthan twofold in the normal tissue is defined as overexpression.

3. The Relation is Validated Between DSG3 Overexpression and ClinicalMalignant Degree in HNC

To compare normal tissues with tumor tissues from 56 patients with HNC,34 patients are found that DSG3 expression in tumor tissue is higherthan in normal tissue and the ratio is up to 61%. Moreover, theassociation of clinicopathologic features and DSG3 expression isexamined by a statistical analysis and significant correlations arefound between DSG3 overexpression and T stage (P=0.009), N stage(P=0.047), overall stage (P=0.011), tumor depth (P=0.009) andextracapsular spread in lymph nodes (P=0.044) (as shown in FIG. 11),wherein DSG3 overexpressions on T stage and overall stage suggest thatDSG3 participate in cancer growth and DSG3 overexpressions on N stage,tumor depth and extracapsular spread in lymph nodes suggest that DSG3participate in cancer invasion. Accordingly, the present inventionvalidates that DSG3 expression is relative to the clinical malignantdegree in HNC and DSG3 overexpression is able to be used on the clinicalapplication for determining the malignant degree of cancer or be themolecular target on the therapy of HNC.

B. Method for Providing a RNA Jamming Sequence on Inhibition-Specific ofDSG3 Expression

1. Mechanism of RNA Interference

The mechanism of RNA interference is that a little clone of RNA (about17˜22 pieces of Nucleotide) combines with message RNA (mRNA) in cellsthrough sequence matching, is identified by protein Dicer in cells,attracts RISC enzyme, and then the RNA is decomposed to accomplish theinhibition-specific of DSG3 expression. However, the inhibition effectof RNA interference greatly depends on the sequence of the little cloneof RNAi transfected into the cells, and the present invention finds aclone of sequence-specific (DSG3-RNAi) in DSG3 molecules that is capableof inhibiting DSG3 expression effectively.

2. Design Process and Method of DSG3-RNAi

The inhibition of DSG3 reduces cell growth and colony formation by theinterference of a clone of sequence, and the clone of sequence is namedDSG3-RNAi. The DSG3-RNAi is situated from the 2761st Nucleotide towards3′-downstream to the 18th Nucleotide in DSG3 mRNA and has the sequenceof 5′-TTGTTAAGTGCCAGACTT-3′ (SEQ. ID. NO. 2) (nucleotide 660-677) to acton DSG3 mRNA. The experiment method is as follows:

(1) Two Clones of DSG3-RNAi Sequences is Synthesized in the Structure ofSymmetrical Complementary Sequence (Nucleotide 656-707)

(SEQ. ID. NO. 3) 5′-GGATTTGTTAAGTGCCAGACTTgaagcttgAAGTCTGGCACTTAACAAATCC-3′ (SEQ. ID. NO. 4)5′-GGATTTGTTAAGTGCCAGACTTcaagcttcAAGTCTGGCACTTAACA AATCC-3′(2) The Synthesized Sequence is Linked into an Expression Vector

The two clones of synthesized Nucleotide RNAi sequences are heatedindividually at 95° for 10 min and cooled slowly at room temperature forthe matching of the two clones of synthesized sequences, and then theexpression vector is cleaved by using restriction enzyme and is insertedthe two clones of RNAi sequences individually to form DSG3-RNAi plasmidby using T4 DNA ligase assembly. The assembled sequence expresses incoliform and the DSG3-RNAi plasmid is extracted for differentapplications.

3. DSG3-RNAi is Validated on Inhibition-specific of DSG3 Expression

The DSG3-RNAi plasmid and a vector (hereinafter, inscribed as “vectorcontrol”) that has no suppression sequence are transfected into celllines, and DSG3-RNAi plasmid forms a special hairpin structure in celllines after transcription (as shown in FIG. 2).

Effects of DSG3-RNAi on DSG3 expression in HNC cell lines such as OEC-M1and BM1 are analyzed by Western blot analysis to compare with DSG3expression in a vector control 50 through extracting proteins in celllines. Referring to FIGS. 3A and 3B that show the results after 2 days,the results indicate DSG3 expression is inhibited in DSG3-RNAi for 4days. An actin protein expression serves as an internal control andthere is no effect of DSG3-RNAi on actin expression as shown in FIGS. 3Aand 3B. The results validate that DSG3-RNAi sequence is able to beinhibition-specific on DSG3 expression.

C. DSG3-RNAi is Specifically Inhibiting the Malignant Proliferation andInvasion Migration of Cancer Cells

1. DSG3 Knockdown Inhibits Cell Growth in HNC Cell Lines.

DSG3-RNAi sequence-specific is transfected into cancer cell lines andeffects of DSG3-RNAi on cell growth in HNC cell lines are observed tocompare with the vector control. The variation of cell growth isobserved by counting cell numbers for 4 days, results are shown in FIGS.4A and 4B. Referring to FIGS. 4A and 4B, the results indicate thatknockdown of DSG3 expression inhibits cell growth in HNC cell lines.

2. DSG3 Knockdown Inhibits Colony Formation in HNC Cell Lines.

Cellular colony formation is determined by colony formation assay, andthe assay is investigated and observed by photograph observation andquantitative colony numbers as shown in FIG. 5A, and FIG. 5B isquantitative determination of colony numbers on colony formation. In theexperiments on oral carcinoma cells (OECM1) and nasopharymneal carcinomacells (BM1) respectively, the colony numbers and size of carcinoma cellsare less and smaller than vector control after transfection withDSG3-RNAi and various numbers of cells (300-1000 for OECM1 and BM1) areseeded. Referring to FIG. 5A, the colony numbers and size of carcinomacells are less and smaller than vector control after DSG3 expression isinhibited by DSG3-RNAi, and 500 cells are seeded for quantitative assayand results are shown in FIG. 5B that is quantitative assay of cellularcolony formation. In the experiments on OECM1 and BM1 respectively, thecolony numbers and size of carcinoma cells are less and smaller thanvector control after transfection with DSG3-RNAi and 500 cells for OECM1and BM1 are seeded. The foregoing both two kinds of experiments validatethat knockdown of DSG3 expression inhibits cell growth in HNC celllines.

3. DSG3 Knockdown Inhibits Cell Migration in HNC Cell Lines

After DSG3-RNAi is transfected into HNC cell lines, cell migration isdetermined using in vitro wound healing assays to observe and compareeffects of cell migration and invasion with vector control as shown inFIG. 6. In the experiments on OECM1 and BM1 in vitro wound healingassays respectively, cells are wounded by a micropipette tip and cellmigration toward the wounded is observed. Referring to FIG. 6, DSG3-RNAiis able to inhibit cell migration compared with vector control in OECM1and BM1 cell lines

4. DSG3 Knockdown Inhibits Cell Invasion in HNC Cell Lines.

Similarly, cell invasion is determined using in vitro Matrigel Transwellinvasion assays to analyze and quantify the penetration ability of cellsby counting the cell numbers that penetrate Matrigel for 5 days and theresults show in FIGS. 7A and 7B. In the experiments on OECM1 and BM1 invitro Matrigel Transwell invasion assays respectively, cell invasion isdetermined by observing cell numbers that penetrate Matrigel for 5 daysand the results indicate that DSG3-RNAi apparently inhibits the abilityof cell invasion compared with vector control in OECM1 and BM1 celllines as shown in FIGS. 7A and 7B. In FIGS. 7A and 7B, the numbers ofcarcinoma cells that penetrate Matrigel are less than vector controlafter DSG3 expression is inhibited by DSG3-RNAi and the foregoing bothtwo kinds of experiments validate that knockdown of DSG3 expressioninhibits the abilities of cell migration and invasion in HNC cell lines.

5. DSG3 Knockdown Inhibits Cancer Cell Growth In Vivo.

The experiment is to validate that DSG3 knockdown inhibits cancer cellgrowth in vivo animal and the BALB/C-nu nude mice at 5 weeks of age areused for the experiment. The mice are subcutaneously injected with 10⁷cancer cell lines and are intravenously injected 50 μg of control vectoror DSG3-RNAi plasmid after 3 days, followed by a booster of 25 μg ofvector or plasmid twice a week for a total 6 weeks. Each treatment grouphas five mice, the cancer cells are monitored for a total of 60 days andthen the cancer cell volume is calculated as length×width×height andresults are shown in FIG. 8A. In vector control, the cancer cell growthis continuous, but DSG-RNAi apparently inhibits the cancer cell growth.Besides, six weeks after grafting, cancer cells are removed and observedon DSG3 expression and hair follicle tissues were also stained asinternal control. The results validate that DSG3 expression is certainlyinhibited by DSG3-RNAi (as shown in FIG. 8B), and that DSG3 knockdowninhibits cancer cell growth is validated.

6. DSG3 Knockdown Inhibits Cancer Cell Migration In Vivo.

The experiment is to validate that DSG3 knockdown inhibits cancer cellmigration in vivo animal and the BALB/C-nu nude mice at 5 weeks of ageare used for the experiment. The mice are intravenously injected with5×106 cancer cell lines so as to simulate that cancer cells flow intoblood and observe if cancer cells migrate to liver and are intravenouslyinjected 50 μg of control vector or DSG3-RNAi plasmid after 3 days,followed by a booster of 25 μg of vector or plasmid twice a week for atotal 3 weeks. The vector control group has seven mice and theexperimental group has six mice, the cancer cells are monitored for atotal of 30 days and then the livers are took out by sacrificing themice to be observed and determined the hepatic migration. Referring toFIG. 9A, there are a plurality of yellowish-white cancer cells at theliver in vector control group, and there is no hepatic migration in theexperimental group that using reagents. The statistical table is shownin FIG. 9B that six mice display hepatic migration (85.7%) in vectorcontrol group and no mouse displays hepatic migration (0%) inexperimental group. The results validate that DSG3 Knockdown inhibitscancer cell migration.

Known by the aforesaid, the present invention provides a method forclinical or prognosis applications on determining the malignant degreeof HNC including tumor size, Lymph invasion, tumor depth and cancerstage by determining DSG3 expression. The present invention alsoprovides a RNA jamming sequence, DSG3-RNAi sequence, oninhibition-specific of DSG3 expression for inhibiting DSG3 expressionand then inhibiting cell growth and invasion in HNC. Therefore, theDSG3-RNAi sequence is applied on the development of cancer medicine, theRNAi that act on DSG3 mRNA is 5′-TTGTTAAGTGCCAGACTT-3′ SEQ. ID. NO. 2.

A method for providing a RNA jamming sequence on inhibition-specific ofDSG3 expression to inhibit the growth of cancer cells, comprising thesteps of:

-   -   (a) designing the jamming sequence, named DSG3-RNAi, so as to        interfere DSG3 expression, wherein the DSG3-RNAi is situated        from the 2761st Nucleotide towards 3′-downstream to the 18th        Nucleotide in DSG3 mRNA and is 5′-TTGTTAAGTGCCAGACTT-3′; (SEQ.        ID. NO. 2) (nucleotide 660-677)    -   (b) synthesizing two clones of DSG3-RNAi sequences in the        structure of symmetrical complementary sequence, such as

(SEQ. ID. NO. 3) 5′-GGATTTGTTAAGTGCCAGACTTgaagcttgAAGTCTGGCACTTAACAAATCC-3′ (SEQ. ID. NO. 4)5′-GGATTTGTTAAGTGCCAGACTTcaagcttcAAGTCTGGCACTTAACA AATCC-3′; (nucleotide656-707)

-   -   (c) linking the synthesized sequence into an expression vector        by the ways of matching the two clones of Nucleotide RNAi        sequences, cleaving the expression vector via using restriction        enzyme, inserting the two clones of RNAi sequences into the        expression vector respectively to assemble and form DSG3-RNAi        plasmid, and extracting the DSG3-RNAi plasmid via expressing the        assembled sequence in coliform;    -   (d) transfecting the DSG3-RNAi plasmid into cell lines so as to        form a special hairpin structure in cell lines after        transcription; and    -   (e) transfecting the DSG3-RNAi plasmid into cancer individuals        respectively for inhibiting cell growth of cancer.

A suppression reagent that is a DSG3-RNAi plasmid for injecting intocancer individuals, wherein the DSG3-RNAi plasmid is a DSG3-RNAisequence and the DSG3-RNAi sequence is 5′-TTGTTAAGTGCCAGACTT-3′ (SEQ.ID. NO. 2) (nucleotide 660-677) to synthesize two clones of DSG3-RNAisequences in the structure of symmetrical and then link the synthesizedsequence into an expression vector.

Although the present invention has been explained in relation to itspreferred embodiments, it is to be understood that many other possiblemodifications and variations can be made without departing from thespirit and scope of the invention as hereinafter claimed.

1. A suppression reagent that is a DSG3-RNAi plasmid for injecting intocancer individuals, wherein the DSG3-RNAi plasmid is a DSG3-RNAisequence and the DSG3-RNAi sequence that act on DSG3 mRNA is5′-TTGTTAAGTGCCAGACTT-3′(SEQ. ID. NO. 2) to synthesize two clones ofDSG3-RNAi sequences in the structure of symmetrical and then link thesynthesized sequence into an expression vector.